Herpes Simplex Virus 1 (HSV-1) and HSV-2 are related human 1-herpesviruses that share the ability to establish long-term latent infections in neurons of primary sensory ganglia. Like other herpesviruses, HSV-1 and HSV-2 express virally encoded microRNAs (miRNAs) in latently infected cells, but these two viruses are unusual in that they do not express any viral proteins during latency, thus suggesting that viral miRNAs are likely to play a key role in the establishment and maintenance of the latent state. Using deep sequencing, we have identified eight miRNAs expressed by HSV-1 and by HSV-2 in latently infected primary neurons in vivo, six of which are evolutionarily conserved between these two viruses in terms of genomic location and primary sequence. Here, we propose extending these in vivo data by using deep sequencing to identify HSV-1 and HSV-2 miRNAs that are expressed in cultured cells productively infected with vhs-deficient virus mutants, to minimize the mRNA fragmentation seen with wildtype HSV-1 or HSV-2. We will then use a combination of computational techniques, mRNA microarray analyses and protein:RNA crosslinking approaches to identify cellular and viral mRNAs that are downregulated by HSV-1 or HSV-2 miRNAs expressed either ectopically, from a lentiviral expression vector, or naturally, from the infecting HSV-1 or HSV-2 genome. Candidate mRNA targets will then be further characterized using indicator assays, Northern and Western blot analyses and, where possible, phenotypic assays. We also intend to examine whether the overexpression of virally encoded miRNAs, or the inhibition of viral miRNA function using antagomirs or miRNA "sponges", affects viral replication potential in culture. Together, these studies not only have the potential to shed significant light on the possibly crucial role of ?-herpesvirus miRNAs in regulating viral latency and pathogenesis but may also suggest novel approaches to the treatment of the diseases induced by these human pathogens.